Inhibitors of Hemeprotein-Catalyzed Lipid Peroxidation

ABSTRACT

Methods and compounds for the treatment or prevention of oxidative damage in a mammalian subject. The treatment and/or prevention may be on inhibiting heme-induced lipid peroxidation. Also discloses are methods and compounds for treating or preventing isoprostane-mediated tissue damage.

PRIORITY INFORMATION

This application is a continuation of U.S. patent application Ser. No.13/759,987, now U.S. Pat. No. 9,133,212, filed Feb. 5, 2013; which is acontinuation-in-part of U.S. patent application Ser. No. 12/056,245, nowU.S. Pat. No. 8,367,669, filed Mar. 28, 2008; which is acontinuation-in-part of U.S. patent application Ser. No. 11/153,134,filed Jun. 15, 2005, now abandoned; and claims benefit to U.S. PatentApplication No. 60/908,185, filed Mar. 26, 2007. The content of allapplications are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made in part with funding provided by grant numbersGM015431 and GM042056 by the National Institutes of Health. The UnitedStates Government has certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates, generally, to the field ofhemeprotein-catalyzed lipid peroxidation, and methods of inhibiting theformation of hemo-protein and heme-mediated oxidation products andmethods for preventing tissue and organ damage associated withhemoprotein and heme-medaited oxidation and oxidation products.

Additionally, the present invention relates to the field of COX-1 andCOX-2 inhibition, and methods of treating cyclooxygenase mediatedindications and/or diseases, including the treatment or alleviation ofinflammation and other inflammation associated disorders such asarthritis and neurodegeneration.

BACKGROUND OF THE INVENTION

Oxidative stress has been associated with a number of disease states,including cardiovascular disorder disorders, neurological disorders,cancer, and diabetes. Heme acts a a pro-inflammatory molecule involvedin the pathology of conditions as diverse as renal failure,arteriosclerosis, and peritoneal endometriosis.

Although correlations have been found between lipid peroxidation and awide variety of seemingly diverse diseases, and certain oxidized lipidshave also been proposed as markers, which would indicate the presence ofor level of oxidative damage, it would be of great value to identify thebiochemical processes that produce such oxidative damage and to identifypharmaceutical agents that may prevent it.

Cyclooxygenase is an enzyme that catalyzes a rate-determining step inthe biosynthesis of prostaglandins, which are important mediators ofinflammation and pain. The enzyme occurs as at least two distinctisomers, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). TheCOX-1 isomer is constitutively expressed in the gastric mucosa,platelets and other cells and is involved in the maintenance ofhomeostasis in mammals, including protecting the integrity of thedigestive tract. The COX-2 isomer, on the other hand, is notconstitutively expressed but rather is induced by various agents, suchas cytokines, mitogens, hormones and growth factors. In particular,COX-2 is induced during the inflammatory response (DeWitt D L, BiochimBiophys Acta, 1083:121-34, 1991; Seibert et al., Receptor, 4:17-23,1994.). Aspirin and other conventional non-steroid anti-inflammatorydrugs (NSAIDs) are non-selective inhibitors of both COX-1 and COX-2.They can be effective in reducing inflammatory pain and swelling, butthey produce undesirable side effects of gastrointestinal pathology.

Thus, NSAIDs are widely used in treating pain and the signs and symptomsof arthritis because of their analgesic and anti-inflammatory activitybecause of the acceptance that they work by blocking the activity ofcyclooxygenase (COX), also known as prostaglandin G/H synthase (PGHS),the enzyme that converts arachidonic acid into prostanoids.Prostaglandins, especially prostaglandin E₂ (PGE₂), which is thepredominant eicosanoid detected in inflammation conditions, aremediators of pain, fever and other symptoms associated with inflammationInhibition of the biosynthesis of prostaglandins has been a therapeutictarget of anti-inflammatory drug discovery. The therapeutic use ofconventional NSAIDs is, however, limited due to drug associated sideeffects, including life threatening ulceration and renal toxicity. Analternative to NSAIDs is the use of corticosteriods, however, long termtherapy can also result in severe side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that shows inhibition of PGHS-1 by N-dimethylpyridinol and N-dimethyl pyrimidinol analogues of acetaminophen (ApAP).

FIG. 2 is a graph that shows the effect of ApAP and DM-Pym analog on theinitial rate of reduction of ferryl-myoglobin.

FIG. 3 is a graph that shows the selective inhibition of COX isoforms byacetaminophen analogs of the present invention.

FIG. 4 is a graph that shows cytotoxicity of acetaminophen analogs ofthe present invention in HepG2 cells.

FIG. 5 is a graph that shows the effect of ethanol on cytotoxicity ofacetaminophen analogs in HepG2 cells.

DESCRIPTION OF THE INVENTION

As indicated above, the presently disclosed invention disclosescompounds, compositions, and methods of using said compounds andcompositions.

With respect to the present invention, the following terms are believedto be well understood by one of ordinary skill in the art, the followingdefinitions are set forth to facilitate explanation of the presentlydisclosed subject matter.

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. References to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques that would be apparent to one of skill in the art.While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Following long-standing patent law convention, the terms “a”, “an”, and“the” mean “one or more” when used in this application, including theclaims. Thus, for example, the phrase “a reactive oxygen species” refersto one or more reactive oxygen species.

The term “about”, as used herein when referring to a measurable valuesuch as an amount of weight, time, dose (e.g., a dose of a compound ofthe present invention), etc., is meant to encompass variations of insome embodiments +/−20%, in some embodiments +/−10%, in some embodiments+/−5%, in some embodiments 1%, and in some embodiments +/−0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed methods.

As used herein, the term “and/or” refers to alternatives in which one ormore of the listed entities is present. For example, the phrase “Aand/or B” refers to alternatives wherein A is present, B is present, orboth A and B are present. In those cases where more than twoalternatives are present, the phrase “and/or” refers to alternatives inwhich any one of the listed entities is present, all of the listedentities are present, or any subset of listed entities is present.

As used herein, the phrase “associated with” refers to a relationshipbetween two or more occurrences that one of ordinary skill in the artwould recognize is normally or frequently observable when one or more ofthe occurrences is present. For example, a “symptom associated with adisorder in a subject” is a symptom that is normally, frequently, orsometimes present in the subject when the subject has the disorder. Itis understood, however, that the symptom need not necessarily beindicative of the disorder, causative of the disorder, or absent in thesubject in the absence of the disorder. Thus, the phrase “associatedwith” does not necessarily imply a causal relationship between the twoor more occurrences, although in some embodiments a causal relationshipcan exist.

For example, in some embodiments the phrase “kidney disorder that isassociated with oxidative stress, carbonyl stress, or combinationsthereof” refers to any nephropathy at least one symptom of which iscaused by or modulated by oxidative stress, carbonyl stress, orcombinations thereof, as those terms would be understood by one ofordinary skill in the art after review of the instant disclosure. Insome embodiments, a “kidney disorder that is associated with oxidativestress, carbonyl stress, or combinations thereof” is a medical conditionassociated with elevated levels of reactive carbonyl species (RCS),reactive oxygen species (ROS), and/or advanced glycation end products(AGE). In some embodiments, a “kidney disorder that is associated withoxidative stress, carbonyl stress, or combinations thereof” comprisesacute renal injury (ARI), acute renal failure (ARF), and combinationsthereof.

As used herein with respect to compositions comprising compounds of thepresent invention, the phrase “effective amount” refers to an amount ofcompounds and/or compositions of the present invention that whenadministered to a subject as a single dose or in multiple doses leads toan amelioration of (e.g., an improvement of, a decreased duration of,etc.) at least one symptom of a disorder disclosed herein. In someembodiments, the disorder and/or the symptom is associated withoxidative stress, carbonyl stress, or combinations thereof in thesubject. In some embodiments, the effective amount reduces formation of,reactivity of, or both formation and reactivity of at least one RCS,ROS, or AGE in order to ameliorate at least one symptom of the diseaseassociated with oxidative stress, carbonyl stress, or combinationsthereof in the subject.

Oxidative stress is the term used to describe a physiological state thatcan promote and/or can be associated with an increase in the level ofreactive oxygen species (ROS) and reactive nitrogen species (NOS),either from injury or disease processes, or a decrease in endogenousprotective anti-oxidative capacity, or both. Oxidative stress is usuallyaccompanied by carbonyl stress characterized by an increase inproduction of low molecular weight reactive carbonyl species (RCS). Inmany types of illnesses, including but not limited to sepsis, trauma,burn injury, acute pancreatitis, liver injury, severe diabetes, acuterespiratory distress syndrome, AIDS, and acute renal failure, increasedoxidative stress and/or carbonyl stress can occur.

As used herein, the term “treatment”, and grammatical variants thereof,refers to a medical intervention that is designed to reduce or eliminateat least one symptom resulting from a disease process as describedherein. The term “prevention”, and grammatical variants thereof, refersto a medical intervention that is designed to retard or prevent theinitial development or subsequent progression of at least one symptomresulting from a disease process as described herein. Thus, in someembodiments “prevention” and “treatment” can overlap. As such, the termsare used substantially interchangeably herein, although it is understoodthat “treatment” implies that at least one symptom resulting from adisease process as disclosed herein has become manifest in someobservable and/or quantifiable fashion.

In some embodiments, the methods disclosed herein provide for treatmentand/or prevention of acute renal failure and/or acute renal injury in asubject. As used in this context, the term “prevent” is also intended torelate to a prophylactic approach, such that “preventing” includes bothmodulating the initial development of a disease process as well asmodulating the further development of (i.e., the worsening of) a diseaseprocess. It is understood that the degree of prevention/prophylaxis neednot be absolute (e.g., complete prevention of the development of adisease process such that the subject does not develop the diseaseprocess at all), and that intermediate levels of prevention/prophylaxisincluding, but not limited to increasing the time required for at leastone symptom resulting from a disease process to develop, reducing theseverity of at least one symptom resulting from a disease process, andreducing the time that at least one symptom resulting from a diseaseprocess is present within the subject are all examples ofprevention/prophylaxis. With respect to the latter two circumstances,these are examples wherein “prevention/prophylaxis” and “treatment” canbe considered to coincide.

It is also understood that the disclosed methods can be used as part ofa combination therapy, and need not be employed as the sole therapy toaddress a disease process as disclosed herein.

As used herein, the term “alkyl” means C₁₋₁₂, inclusive (i.e., carbonchains comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms;also, in some embodiments, C₁₋₆ inclusive) linear, branched, or cyclic,saturated or unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains,including for example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, pentyl, hexyl, ethenyl, propenyl, butenyl,pentenyl, hexenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, andallenyl groups.

The alkyl group can be optionally substituted with one or more alkylgroup substituents which can be the same or different, where “alkylgroup substituent” includes alkyl, halo, aryl, arylamino, acyl, hydroxy,aryloxy, alkoxyl, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy,alkoxycarbonyl, oxo, and cycloalkyl. In this case, the alkyl can bereferred to as a “substituted alkyl”. Representative substituted alkylsinclude, for example, benzyl, trifluoromethyl, and the like. There canbe optionally inserted along the alkyl chain one or more oxygen, sulfuror substituted or unsubstituted nitrogen atoms, wherein the nitrogensubstituent is hydrogen, alkyl (also referred to herein as“alkylaminoalkyl”), or aryl. Thus, the term “alkyl” can also includeesters and amides. “Branched” refers to an alkyl group in which an alkylgroup, such as methyl, ethyl, or propyl, is attached to a linear alkylchain.

Administration can be by any method known to one of ordinary skill inthe art. In some embodiments, suitable methods for administration ofcompounds of the present invention include, but are not limited tointravenous administration, bolus injection, and oral administration.

An effective dose for use in the presently disclosed methods isadministered to a subject in need thereof. Thus, in addition to above,the phrase “effective amount” can also refer to an amount of atherapeutic composition of the present invention sufficient to produce abiologically or clinically relevant response (e.g., a “benefit”) in asubject being treated. The actual amount delivered can be varied so asto administer an amount that is effective to achieve the desiredtherapeutic response for a particular subject.

The potency of a composition can vary, and therefore an “effectiveamount” can vary. However, using standard assay methods, one skilled inthe art can readily assess the potency and efficacy of the presentinvention, and adjust the therapeutic regimen accordingly.

After review of the disclosure of the presently disclosed subject matterpresented herein, one of ordinary skill in the art can tailor thedosages to an individual subject, taking into account the particularformulation, method of administration to be used with the composition,and particular disease process to be treated and/or prevented. Furthercalculations of dose can consider subject height and weight, severityand stage of symptoms, and the presence of additional deleteriousphysical conditions. Such adjustments or variations, as well asevaluation of when and how to make such adjustments or variations, arewell known to those of ordinary skill in the art of medicine. In someembodiments, the effective amount is selected from the group consistingof less than 1 mg/day, about 1-10 mg/day, about 10-50 mg/day, about50-100 mg/day, about 100-200 mg/day, about 200-300 mg/day, about 300-400mg/day, about 400-500 mg/day, and more than 500 mg/day.

As is known in the art, these dosages can be administered at one time oras part of two or more daily administrations. For example, for oraladministration, the dose can be in some embodiments about 50 mg/dose bidin die (BID), in some embodiments about 100 mg/dose BID, and in someembodiments about 250 mg/ml BID. For intravenous administration, thedaily dose can be in some embodiments about 25 mg/day, in someembodiments about 50 mg/day, and in some embodiments about 200 mg/day.It is understood that the effective amount might vary among patients,and further that the actual dose administered can easily be modified bya physician as needed.

One of ordinary skill in the art will appreciate that the compounds ofthe invention are useful in treating a diverse array of diseases. One ofordinary skill in the art will also appreciate that when using thecompounds of the invention in the treatment of a specific disease thatthe compounds of the invention may be combined with various existingtherapeutic agents used for that disease.

The compounds of the invention can also be used in combination withexisting therapeutic agents for the treatment of osteoarthritis.Suitable agents to be used in combination include standard non-steroidalanti-inflammatory agents (hereinafter NSAID's) such as piroxicam,diclofenac, propionic acids such as naproxen, flurbiprofen, fenoprofen,ketoprofen and ibuprofen, fenamates such as mefenamic acid,indomethacin, sulindac, apazone, pyrazolones such as phenylbutazone,salicylates such as aspirin, COX-2 inhibitors such as celecoxib androfecoxib, analgesics and intraarticular therapies such ascorticosteroids and hyaluronic acids such as hyalgan and synvisc.

The active ingredient of the present invention may be also administeredin combination with inhibitors of other mediators of inflammation,comprising one or more members selected from the group consistingessentially of the classes of such inhibitors and examples thereof whichinclude, matrix metalloproteinase inhibitors, aggrecanase inhibitors,TACE inhibitors, IL-1 processing and release inhibitors, ILra,H₁-receptor antagonists; kinin-B₁- and B₂-receptor antagonists;prostaglandin inhibitors such as PGD-, PGF-PGI₂2-, and PGE-receptorantagonists; thromboxane A₂ (TXA2-) inhibitors; 5- and 12-lipoxygenaseinhibitors; leukotriene LTC₄-, LTD₄/LTE₄-, and LTB₄-inhibitors;PAF-receptor antagonists; gold in the form of an aurothio group togetherwith various hydrophilic groups; immunosuppressive agents, e.g.,cyclosporine, azathioprine, and methotrexate; anti-inflammatoryglucocorticoids; penicillamine; hydroxychloroquine; anti-gout agents,e.g., colchicine, xanthine oxidase inhibitors, e.g., allopurinol, anduricosuric agents, e.g., probenecid, sulfinpyrazone, and benzbromarone.

The compounds of the present invention may also be used in combinationwith anticancer agents such as endostatin and angiostatin or cytotoxicdrugs such as adriamycin, daunomycin, cis-platinum, etoposide, taxol,taxotere and alkaloids, such as vincristine, and antimetabolites such asmethotrexate.

The compounds of the present invention may also be used in combinationwith anti-hypertensives and other cardiovascular drugs intended tooffset the consequences of atherosclerosis, including hypertension,myocardial ischemia including angina, congestive heart failure, andmyocardial infarction, selected from diuretics, vasodilators such ashydralazine, β-adrenergic receptor antagonists such as propranolol,angiotensin-II converting enzyme inhibitors (ACE-inhibitors) such asenalapril used to treat geriatric mammals with mitral insufficiency, andenalapril alone and in combination with neutral endopeptidaseinhibitors, angiotensin II receptor antagonists such as losartan, renininhibitors, calcium channel blockers such as nifedipine, α₂-adrenergicagonists such as clonidine, α-adrenergic receptor antagonists such asprazosin, and HMG-CoA-reductase inhibitors (anti-hypercholesterolemics)such as lovastatin or atorvastatin.

The active ingredient of the present invention may also be administeredin combination with one or more antibiotic, antifungal, antiprotozoal,antiviral or similar therapeutic agents.

The compounds of the present invention may also be used in combinationwith CNS agents such as antidepressants (such as sertraline),anti-Parkinsonian drugs (such as L-dopa, requip, miratex, MAOBinhibitors such as selegine and rasagiline, comP inhibitors such asTasmar, A-2 inhibitors, dopamine reuptake inhibitors, NMDA antagonists,nicotine agonists, dopamine agonists and inhibitors of neuronal nitricoxide synthase), and anti-Alzheimer's drugs such as donepezil, tacrine,COX-2 inhibitors, propentofylline or metryfonate.

The compounds of the present invention may also be used in combinationwith osteoporosis agents such as roloxifene, droloxifene or fosomax andimmunosuppressant agents such as FK-506 and rapamycin.

This invention also relates to method for treating or preventingdiseases or conditions mediated by cyclooxygenase-2 in a mammalcomprising administering an amount of a compound according to claim 1 ora pharmaceutically acceptable salt thereof effective for treating saiddiseases or conditions to said mammal.

This invention also relates to a pharmaceutical composition comprisingan amount of a compound of the present invention or a pharmaceuticallyacceptable salt thereof effective for treating or preventing diseases orconditions mediated by cycloxygenase-2.

The present invention also relates to the inhibition ofmyoglobin-induced lipid peroxidation, such as occurs in rhabdomyolysis.

The present invention also relates to the inhibition ofmyeloperoxidase-induced lipid peroxidation. Myeloperoxidase-inducedlipid peroxidation and protein modification are thought to be importantcontributors to atherosclerotic plaques

More specifically, this invention relates to a pharmaceuticalcomposition for treating a disease, or condition selected from the groupconsisting of diseases or conditions in which prostaglandins areimplicated as pathogens, pain, fever, inflammation, rheumatic fever,symptoms associated with influenza and other viral infections, commoncold, low back and neck pain, dysmenorrhea, headache, toothache, sprainsand strains, myositis, neuralgia, synovitis, arthritis includingrheumatoid arthritis, degenerative joint disease or osteoarthritis, goutand ankylosing spondylitis, bursitis, burns, injuries following surgicaland dental procedures, disease or conditions associated with cellularneoplastic transformations and metastic tumor growth, cancer, colorectalcancer, breast and skin cancer, familiar adenomatous polyposis,cyclooxygenase-mediated proliferation disorders, cyclooxygenase-mediatedproliferation disorders in diabetic retinopathy and tumor angiogenesis,prostaniod-induced smooth muscle contraction mediated by synthesis ofcontractile prostanoids, dysmenorrhea, premature labor, asthma,eosinophil related disorders, neurodegenerative diseases, Alzheimer'sand Parkinson's disease, bone loss, osteoarthritis, peptic ulcers,gastritis, regional enterotis, ulcerative colitis, diverticulitis,recurrent of gastrointestinal lesions, gastrointestinal bleeding,coagulation, anemia, hypoprothrombinemia, haemophilia, bleedingproblems; kidney disease and conditions prior to surgery of taking ofanticoagulants.

This invention also relates to a pharmaceutical composition comprisingan amount of a compound of the present invention or a pharmaceuticallyacceptable salt thereof effective for inhibiting hemeprotein-catalyzedlipid peroxidation.

Acetaminophen, with selective analgesic and anti-pyretic effects, isamong the most widely used drugs in the world. Yet there is growingconcern for the hepatotoxicity of the drug and toxicologists in the U.K.predict that the drug would be removed from that market in the event ofa safe and effective alternative.

Acetaminophen acts by blocking biosynthesis of prostaglandinsselectively in cells that are in the signaling pathway for fever andpain. The prostaglandin H synthases (PGHS; cyclooxygenases) thatcatalyze formation of the prostaglandins are bifunctional enzymes withperoxidase and cyclooxygenase active sites. Reduction of a hydroperoxidein the peroxidase site generates a protoporphyrin radical that leads toformation of a tyrosyl radical in the cyclooxygenase site. This tyrosylradical then initiates the oxygenation of arachidonic acid that resultsin prostaglandin formation. This enzyme, therefore, functionsessentially as a hemoprotein that catalyzes structurally specific lipidoxygenation. Acetaminophen has been known to reduce the PGHSprotoporphyrin radical, and our recent work has demonstrated thatacetaminophen acts to inhibit prostaglandin formation selectively incells in which the concentration of hydroperoxides is low enough topermit acetaminophen to maintain the enzyme in a reduced andcatalytically inactive state. The compounds of the present invention, asinhibitors of hemoprotein-catalyzed lipid peroxidation are markedly morepotent than acetaminophen as inhibitors of the PGHSs, and cell basedtoxicology studies indicate that lead compounds are likely to be free ofthe hepatotoxicity produced by acetaminophen.

The compounds if the present invention, therefore, are attractive forother important indications.

One embodiment of the present invention relates to the treatment ofsubarachnoid hemorrhage with inhibitors of hemeprotein-catalyzed lipidperoxidation.

Aneurysmal subarachnoid hemorrhage (SAH) is an often devastating form ofstroke with high morbidity and mortality despite advances in surgicalmanagement. Approximately 30,000 patients annually suffer from SAH inthe U.S., and the worldwide annual incidence approaches 400,000. Forpatients who survive the initial subarachnoid hemorrhage, delayedcerebral vasospasm occurring from days 4-14 is the greatest cause ofneurological disability and death.

A growing body of evidence incriminates hemoprotein-catalyzed lipidperoxidation as the mediator of the vasospasm. Hemoglobin released fromlysed red cells in the subarachnoid space becomes oxidized, in whichstate it acts as a pseudoperoxidase and generates the protein radicalsthat induce lipid peroxidation. F₂-isoprostanes formed by this lipidperoxidation are highly potent constrictors of cerebral arterioles. Thepresent inventors have demonstrated a more than 5 fold mean increase inF₂-isoprostanes in the cerebrospinal fluid of patients with SAH; thisincrease is maximal at the time of delayed vasospasm, and the level ofincrease is a function of the severity of the SAH. We hypothesize thatsuch vasoconstrictors are major contributors to the vasospasm producedby hemoglobin in subarachnoid hemorrhage.

The present inventors have discovered that acetaminophen is a potentinhibitor of hemoprotein-catalyzed lipid peroxidation with an IC₅₀ forhemoglobin of 15 μM, which is in the range of plasma levels resultingfrom therapeutic doses of the drug in humans. Acetaminophen acts byreducing the ferryl-oxo radical form of the heme, and thereby preventsformation of the hemoprotein radical that initiates lipid peroxidationby hemoglobin as well as by myoglobin. To assess proof of concept invivo, we determined the effect of acetaminophen in a rat model ofrhabdomyolysis in which renal failure is caused by intense vasospasmresulting from myoglobin-catalyzed lipid peroxidation. Acetaminophenblocked lipid peroxidation in this model, and prevented the renalfailure with a dose that produced plasma levels in the therapeutic rangefor humans.

An inhibitor of hemeprotein-catalyzed lipid peroxidation is consideredfor a number of potential therapeutic targets. These include the renalfailure that results from release of myoglobin from skeletal muscle inrhabdomyolysis and the myocardial reperfusion injury associated withrelease of myoglobin from ischemic cardiomyocytes. Hemoglobin-inducedlipid peroxidation is linked to the pathophysiology of the renal failureassociated with the massive hemolysis in Plasmodium falciparum malariaand of the pulmonary crisis in sickle cell disease, in addition to thevasospasm in subarachnoid hemorrhage.

Subarachnoid hemorrhage has been selected as the initial target foracetaminophen and related inhibitors of hemoglobin-induced lipidperoxidation. This selection is based on the strength of the evidencethat lipid peroxidation contributes to the vasospasm in subarachnoidhemorrhage, on the catastrophic consequences of this disease, andparticularly on the opportunity to initiate pre-emptive therapy inadvance of the delayed vasospasm. The NIH has concurred with this targetselection, and is funding a pilot study to assess the extent ofacetaminophen's effect on lipid peroxidation in patients withsubarachnoid hemorrhage.

Another aspect of the present invention is a strategy for development ofeven more potent inhibitors. We considered that the inhibition couldresult from either electron transfer or hydrogen atom transfer to theprotoporphyrin radical. Acetaminophen (ApAP) is a phenol and as such itcould act as an antioxidant by giving up its phenolic hydrogen atom to achain-propagating lipid peroxyl radical. The bond dissociation enthalpy(BDE) of the phenolic O—H plays a central role in determiningantioxidant efficacy, compounds having lower BDEs generally being betterantioxidants. Electron-rich phenols like ApAP also serve as goodelectron donors, and our findings indicate that electron donation is thelikely mechanism by which this compound inhibits hemoprotein-inducedlipid peroxidation. The ionization potential (IP) of a molecule reportsits ability to donate an electron; the lower the IP, the better theelectron donor. Recent advances in computational chemistry permitexcellent prediction of BDEs and IPs of ApAP and analogs. Computed BDEsare within a kcal/mol or so of experimentally obtained values andcalculated IPs provide excellent comparisons within series of compounds.In Table 1 below are presented the calculated BDEs and IPs of a seriesof phenolic compounds of the present invention that includes ApAP. Whatis striking about these data is that there are a number of structurallyrelated molecules that have substantially lower BDEs and IPs than doesApAP. It is our hypothesis that these compounds will be substantiallybetter H atom and electron donors than is ApAP. As a result, suchcompounds will be better antioxidants in general and better inhibitorsof hemoprotein-induced lipid peroxidation in particular than is ApAP.Our investigations with the cyclooxygenases and myoglobin confirm bothof these predictions.

Another embodiment of the present invention is methods of COX-1 andCOX-2 inhibition, and methods of treating cyclooxygenase mediatedindications and/or diseases, including the treatment or alleviation ofinflammation and other inflammation associated disorders such asarthritis and neurodegeneration.

Thus, embodiments of the present invention include the below compoundsand their methods of use. As one of ordinary skill in the art wouldreadily recognize, the synthesis of the compounds is reasonablystraightforward.

Further examples of compounds of the present invention include thefollowing:

TABLE 1 Additional Embodiments of the Invention BDE  82.5  83.2  83.7 77.0  78.3  73.5  74.1 (kcal/mol) IP 176   184   193   164   175  158   167   (kcal/mol)

ApAP ApPyr ApPym Me₂NPyr Me₂NPym Me₂NDMPyr Me₂NDMPym

The first phase of synthesis and evaluation of additional inhibitors isbased on the premise that success in treating diseases resulting fromhemoprotein-catalyzed lipid peroxidation will require intervention atthe radical initiation step. Thus, aspects of the present inventionfocus on aqueous soluble agents with improved potency in reducing thehemoprotein protoporphyrin radical, building on the advances in potencyalready achieved with the lead compounds in this series.

The process of radical initiation and subsequent propagation is subjectto potential inhibition at multiple steps, and it has dual amplificationmechanisms. It is probably best considered as occurring at anaqueous-lipid phase interface, with the hemoprotein in the aqueousphase, and with hemoprotein radicals eliciting radical formation at theinterface with the lipid phase to initiate chain propagation in thatphase.

Phenolic Antioxidants/Reducing Agents of the Present Invention.

Antioxidants, most commonly substituted phenols, effectively interceptperoxyls by transferring the phenolic H-atom to a propagating peroxylradical, with a rate constant k_(inh) that is faster than that of chainpropagation. The most famous example of a phenolic antioxidant isvitamin E (α-toco-pherol, α-TOH) nature's hydrophobic defense againstradical chain oxidation.

Compounds of the present invention have a higher k_(inh) and serve asbetter antioxidants than α-TOH in chemical tests. The criticalantioxidant step is shown in Equation 1, the larger the k_(inh), thebetter the antioxidant. The simple pyridinol 2a shown below is nearly 5times better than α-TOH as an antioxidant and the bicyclic compounds 3aand 4a are over 20 times better than the vitamin. ApAP, on the otherhand, is a poor antioxidant compared to α-TOH, its rate constant forinhibition being 100-fold less than that of α-TOH.

Relative Potency:

Structural characteristics that make the pyridinol and pyrimidinolcompounds good antioxidants also promise to make them good inhibitors ofhemoprotein induced lipid peroxidation. Antioxidant activity and hemereduction efficacy both depend on the electron donor character ofsubstituents on the phenol. Compounds in the series of pyridinols andpyrimidinols and the bicyclic compounds 4 and 5 are more electron-richthan ApAP and thus they are better antioxidants and promise to be morepotent reductants of reactive heme-oxo species such as O═Fe(IV)PP⁺ thanis ApAP.

Also included in the scope of the present invention are pharmaceuticalcompositions comprising compounds of the present invention, as well astheir methods of use for treating patients in need thereof.

With respect to the compounds of the present invention, the followingcompound,

N-dimethyl-pyrimidinol, especially has been found to have promisingcharacteristics; it is 14 times more potent in reducing ferryl myoglobinthan is acetaminophen, 10 times more potent as an inhibitor of PGHS-1and -2, and it is not cytotoxic in a HepG2 cell line stimulated withethanol in which acetaminophen is toxic.

In another aspect of the present invention, the compounds of the presentinvention are resveratrole, derivatives, and analogs thereof. The term“resveratrol” is intended to mean either the cis-isomer of resveratrol,the trans-isomer of resveratrol, or a mixture of the two isomers. Theterm is also intended to include both the naturally occurring activeagent and the compound as it may be chemically synthesized in thelaboratory.

Resveratrol (3,5,4′-trihydroxystilbene) has been identified as aconstituent not only of grape skins (Soleas et al. (1995) Am. J. Enol.Vitic. 46(3):346-352) but has also been found to be present in groundnuts, eucalyptus, and other plant species. Goldberg et al. (1995), Am.J. Enol. Vitic. 46(2):159-165. A great deal of interest has been focusedon the compound's antifungal activity and its correlation withresistance to fungal infection. Id at 159. Resveratrol may be obtainedcommercially (typically as the trans isomer, e.g. from the SigmaChemical Company, St. Louis, Mo.), or it may be isolated from wine orgrape skins, or it may be chemically synthesized. Synthesis is typicallycarried out by a Wittig reaction linking two substituted phenols througha styrene double bond, as described by Moreno-Manas et al. (1985) Anal.Quim. 81:157-61 and subsequently modified by others (Jeandet et al.(1991) Am. J. Enol. Vitic. 42:41-46; Goldberg et al. (1994) Anal. Chem.66: 3959-63).

There are more studies concerning trans-resveratrol than the cis isomer;however, the cis isomer also appears to be equally important from abiological standpoint. Numerous uses have been proposed and evaluatedfor the resveratrol isomers. Jang et al. (1997) Science 275:218-220,show that resveratrol has cancer chemopreventive activity in assaysrepresenting three major stages of carcinogenesis. That is, the authorsfound that the compound: (1) acted as an antioxidant and antimutagen andinduced phase II drug-metabolizing enzymes (“anti-initiation” activity);(2) mediated anti-inflammatory effects and inhibited cyclooxygenase andhydroperoxidase (“antipromotion” activity); and (3) induced humanpromyelocytic leukemia cell differentiation (“antipromotion” activity).In addition, as noted above, resveratrol has been extensively studiedfor its correlation to the cardiovascular utility of red wine. See,e.g., Bertelli et al., supra; Pace-Asciak et al. (1995), Clinica ChimicaActa 235:207-2191; and Frankel et al. (Apr. 24, 1993), The Lancet341:1104. Neurologic uses have also been proposed (Lee et al. (1994),Society for Neuroscience Abstracts 20(1-2):1648).

Thus, in one embodiment, then, a method is provided for preventing ortreating restenosis in an individual following coronary intervention,comprising treating the individual with a pharmaceutical compositioncomprising a therapeutically effective amount of an active agentselected from the group consisting of resveratrol and pharmacologicallyacceptable salts, esters, amides, prodrugs and analogs thereof.Generally, the active agent will be cis-resveratrol, trans-resveratrol,cis-resveratrol glucoside or trans-resveratrol glucoside, andadministration will be either oral or parenteral. However, as will beappreciated by those skilled in the art, and as discussed in detailelsewhere herein, other forms of the active agents may also be used, asmay a variety of composition types and modes of administration.

In another embodiment, pharmaceutical compositions are provided forcarrying out the present therapeutic method. The compositions contain atherapeutically effective amount of an active agent as described above,and pharmacologically acceptable carrier. Preferably, although notnecessarily, the compositions are oral dosage forms or liquidformulations suitable for parenteral administration, containing theactive agent in unit dosage form.

Thus, the following are compounds of the present invention:

Another embodiment of the present invention includes compounds of thefollowing formula:

Wherein X is N or C, with the C being unsubstituted or substituted withH or alkyl; R₁ is H or alkyl; R₂ is H or alkyl; R₃ is H or alkyl; R₄ isH or alkyl; X₁ is

wherein R is H, alkyl, cycloalkyl, aryl, or —C(O)R; and Y is CH₂, NR, orO; or a pharmaceutically acceptable salt thereof.

Other embodiments of the present invention is compounds of the followingformula:

wherein R₁ is selected from hydrogen, alkyl, C₃₋₁₀ cycloalkyl, aryl,benzyl, heteroaryl, halogen, CN, CF₃, CO, CO-alkyl; R₂ is selected fromhydrogen, alkyl, C₃₋₁₀ cycloalkyl, aryl, benzyl, heteroaryl, halogen,CN, CF₃, CO, CO-alkyl; R₃ is selected from hydrogen, alkyl, C₃₋₁₀cycloalkyl, aryl, benzyl, heteroaryl, halogen, CN, CF₃, CO, CO-alkyl; ora pharmaceutically acceptable salt thereof.

Another embodiment of the present invention includes compounds of thefollowing formula:

wherein X is C, O, or NR₂; R₁ is selected from hydrogen, alkyl, C₃₋₁₀cycloalkyl, aryl, benzyl, heteroaryl, halogen, CN, CF₃, CO, CO-alkyl; R₂is selected from hydrogen, alkyl, C₃₋₁₀ cycloalkyl, aryl, benzyl,heteroaryl, halogen, CN, CF₃, CO, CO-alkyl; or a pharmaceuticallyacceptable salt thereof.

Another embodiment of the present invention is a compound of thefollowing formula:

or a pharmaceutically acceptable salt thereof

With respect to another embodiment of the present invention, thefollowing compound,

2-(dimethylamino)pyrimidin-5-ol (DM-Pym), is also a particularly highlypotent as an inhibitor of PGHS-1 (COX-1) with an IC₅₀ of 21 μM. It ismore than an order of magnitude more potent as a PGHS-1 inhibitor thanis the clinically employed analgesic and anti-pyretic drug,acetaminophen (Tylenol®), which has an IC₅₀ of 250 μM in this in vitrosystem. Additionally, it is equally effective as an inhibitor of PGHS-2(COX-2) whereas its dimethyl-pyridinol analogue is only a weak inhibitorof PGHS-2. This aspect is important therapeutically, as most of thetherapeutic effects of acetaminophen are mediated via its action onPGHS-2.

DM-Pym is also a potent inhibitor of hemoprotein catalyzed lipidperoxidation. the present inventors have demonstrated that acetaminophenis an inhibitor of the lipid peroxidation engendered by thehemoproteins, hemoglobin and myoglobin, in vitro. Moreover, it blocksthe renal lipid peroxidation and renal failure produced byrhabdomyolysis. DM-Pym is remarkably more potent than acetaminophen inreducing ferryl myoglobin and thereby reducing the radical thatinitiates hemoprotein catalyzed lipid peroxidation.

Further, DM-Pym has a more favorable safety profile in cellular toxicitystudies in comparison with acetaminophen. The hepatotoxicity ofacetaminophen is a major problem with its clinical use; not only is thisa cause of liver failure and death from acetaminophen overdose, but italso limits the dose of acetaminophen that can be used for potentialindications such as the diseases caused by hemoprotein catalyzed lipidperoxidation. Acetaminophen-induced hepatotoxicity results from anelectrophilic metabolite that is formed by the catalysis ofacetaminophen by a CYP450 enzyme. Based on this knowledge, a model foracetaminophen toxicity has been developed in which induction of theCYP450 by ethanol in the HepG2 liver cell line makes the cells highlysusceptible to the cytotoxic effect of acetaminophen. In this model, inwhich acetaminophen is shown by us to be cytotoxic, the DM-Pym does notcause cytotoxicity.

A further aspect of the present invention is a biomarker with which totrack clinical development. Measurement of F₂-isoprostanes in thecerebrospinal fluid (CSF) will be employed as a biomarker for lipidperoxidation engendered by subarachnoid hemorrhage and its inhibition.Analysis of F₂-isoprostanes (F₂-IsoPs) by GC/MS has been independentlyvalidated by an NIEHS study to be the most accurate approach forassessing lipid peroxidation and oxidative stress status in vivo. Thuson aspect of the present invention is a highly validated assay toaccurately measure the ability of compounds that will be synthesized toinhibit hemoprotein redox cycling induced lipid peroxidation in vivo.Measurement of F₂-IsoPs is utilized both in animal models ofhemoprotein-induced lipid peroxidation and subsequently in humans totrack clinical development of these compounds. The finding that renalfailure due to rhabdomyolysis is associated with lipid peroxidationreflected by elevated F₂-IsoPs and that it can be inhibited byacetaminophen greatly supports our hypothesis that F₂-IsoPs formed inthe CSF in SAH are key mediators of the vasospasm in SAH. To obtainfurther support for this hypothesis we measured CSF F₂-IsoPs in patientswith SAH. As indicated in the table below, levels of F₂-IsoPs weremarkedly increased in subarachnoid hemorrhage and the extent of increasewas a function of the severity of the subarachnoid bleed (Fisher Grade),and whether the hemorrhage had produced a vegetative state. The highestlevels were seen on day 7 which is the time of delayed vasospasm.

CSF F₂-IsoProstanes (pg/ml) at Peak Value SAH Fisher Grade All III orOutcome = Normal SAH I II III + IV Vegetative state Mean ± 8.7 ± 50.0 ±28.3 39.7 54.9 ± 79.7 ± SE 1.2 5.9 6.8 14.1 (n) (10) (14) (2) (1) (11)(3)

The compounds and compositions of the invention can be administered byany available and effective delivery system including, but not limitedto, orally, bucally, parenterally, by inhalation spray, by topicalapplication, by injection, transdermally, or rectally (e.g., by the useof suppositories) in dosage unit formulations containing conventionalnontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles,as desired. Parenteral includes subcutaneous injections, intravenous,intramuscular, intrasternal injection, or infusion techniques.

Transdermal compound administration, which is known to one skilled inthe art, involves the delivery of pharmaceutical compounds viapercutaneous passage of the compound into the systemic circulation ofthe patient. Topical administration can also involve the use oftransdermal administration such as transdermal patches or iontophoresisdevices. Other components can be incorporated into the transdermalpatches as well. For example, compositions and/or transdermal patchescan be formulated with one or more preservatives or bacteriostaticagents including, but not limited to, methyl hydroxybenzoate, propylhydroxybenzoate, chlorocresol, benzalkonium chloride, and the like.Dosage forms for topical administration of the compounds andcompositions can include creams, sprays; lotions, gels, ointments, eyedrops, nose drops, ear drops, and the like. In such dosage forms, thecompositions of the invention can be mixed to form white, smooth,homogeneous, opaque cream or lotion with, for example, benzyl alcohol 1%or 2% (wt/wt) as a preservative, emulsifying wax, glycerin, isopropylpalmitate, lactic acid, purified water and sorbitol solution. Inaddition, the compositions can contain polyethylene glycol 400. They canbe mixed to form ointments with, for example, benzyl alcohol 2% (wt/wt)as preservative, white petrolatum, emulsifying wax, and tenox II(butylated hydroxyanisole, propyl gallate, citric acid, propyleneglycol). Woven pads or rolls of bandaging material, e.g., gauze, can beimpregnated with the compositions in solution, lotion, cream, ointmentor other such form can also be used for topical application. Thecompositions can also be applied topically using a transdermal system,such as one of an acrylic-based polymer adhesive with a resinouscrosslinking agent impregnated with the composition and laminated to animpermeable backing

Solid dosage forms for oral administration can include capsules,tablets, effervescent tablets, chewable tablets, pills, powders,sachets, granules and gels. In such solid dosage forms, the activecompounds can be admixed with at least one inert diluent such assucrose, lactose or starch. Such dosage forms can also comprise, as innormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, effervescent tablets, and pills, the dosage forms can alsocomprise buffering agents. Soft gelatin capsules can be prepared tocontain a mixture of the active compounds or compositions of theinvention and vegetable oil. Hard gelatin capsules can contain granulesof the active compound in combination with a solid, pulverulent carriersuch as lactose, saccharose, sorbitol, mannitol, potato starch, cornstarch, amylopectin, cellulose derivatives of gelatin. Tablets and pillscan be prepared with enteric coatings.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions can also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

Suppositories for vaginal or rectal administration of the compounds andcompositions of the invention, such as for treating pediatric fever andthe like, can be prepared by mixing the compounds or compositions with asuitable nonirritating excipient such as cocoa butter and polyethyleneglycols which are solid at room temperature but liquid at rectaltemperature, such that they will melt in the rectum and release thedrug.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing agents, wetting agents and/or suspendingagents. The sterile injectable preparation can also be a sterileinjectable solution or suspension in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that can be used are water,Ringer's solution, and isotonic sodium chloride solution. Sterile fixedoils are also conventionally used as a solvent or suspending medium.

The compositions of this invention can further include conventionalexcipients, i.e., pharmaceutically acceptable organic or inorganiccarrier substances suitable for parenteral application which do notdeleteriously react with the active compounds. Suitable pharmaceuticallyacceptable carriers include, for example, water, salt solutions,alcohol, vegetable oils, polyethylene glycols, gelatin, lactose,amylose, magnesium stearate, talc, surfactants, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, and the like. The pharmaceutical preparations canbe sterilized and if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringand/or aromatic substances and the like which do not deleteriously reactwith the active compounds. For parenteral application, particularlysuitable vehicles consist of solutions, preferably oily or aqueoussolutions, as well as suspensions, emulsions, or implants. Aqueoussuspensions may contain substances which increase the viscosity of thesuspension and include, for example, sodium carboxymethyl cellulose,sorbitol and/or dextran. Optionally, the suspension may also containstabilizers.

The composition, if desired, can also contain minor amounts of wettingagents, emulsifying agents and/or pH buffering agents. The compositioncan be a liquid solution, suspension, emulsion, tablet, pill, capsule,sustained release formulation, or powder. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulations can include standard carriers suchas pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, and thelike.

Various delivery systems are known and can be used to administer thecompounds or compositions of the invention, including, for example,encapsulation in liposomes, microbubbles, emulsions, microparticles,microcapsules and the like. The required dosage can be administered as asingle unit or in a sustained release form.

The bioavailabilty of the compositions can be enhanced by micronizationof the formulations using conventional techniques such as grinding,milling, spray drying and the like in the presence of suitableexcipients or agents such as phospholipids or surfactants.

Of course, the compounds and compositions of the invention can beformulated as pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include, for example, alkali metal salts and additionsalts of free acids or free bases. The nature of the salt is notcritical, provided that it is pharmaceutically-acceptable. Suitablepharmaceutically-acceptable acid addition salts may be prepared from aninorganic acid or from an organic acid. Examples of such inorganic acidsinclude, but are not limited to, hydrochloric, hydrobromic, hydroiodic,nitric, carbonic, sulfuric and phosphoric acid and the like. Appropriateorganic acids include, but are not limited to, aliphatic,cycloaliphatic, aromatic, heterocyclic, carboxylic and sulfonic classesof organic acids, such as, for example, formic, acetic, propionic,succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic,glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic,anthranilic, mesylic, salicylic, p-hydroxybenzoic, phenylacetic,mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesuifonic,sulfanilic, stearic, algenic, .beta.-hydroxybutyric,cyclohexylaminosulfonic, galactaric and galacturonic acid and the like.Suitable pharmaceutically-acceptable base addition salts include, butare not limited to, metallic salts made from aluminum, calcium, lithium,magnesium, potassium, sodium and zinc or organic salts made fromprimary, secondary and tertiary amines, cyclic amines,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine and thelike. All of these salts may be prepared by conventional means from thecorresponding compound by reacting, for example, the appropriate acid orbase with the compound.

While individual needs may vary, determination of optimal ranges foreffective amounts of the compounds and/or compositions is within theskill of the art. Generally, the dosage required to provide an effectiveamount of the compounds and compositions, which can be adjusted by oneof ordinary skill in the art, will vary depending on the age, health,physical condition, sex, diet, weight, extent of the dysfunction of therecipient, frequency of treatment and the nature and scope of thedysfunction or disease, medical condition of the patient, the route ofadministration, pharmacological considerations such as the activity,efficacy, pharmacokinetic and toxicology profiles of the particularcompound used, whether a drug delivery system is used, and whether thecompound is administered as part of a drug combination.

The amount of a given compound or composition of the invention that willbe effective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques, including reference to Goodman andGilman, supra; The Physician's Desk Reference, Medical EconomicsCompany, Inc., Oradell, N. J., 1995; and Drug Facts and Comparisons,Inc., St. Louis, Mo., 1993. The precise dose to be used in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided by thephysician and the patient's circumstances.

EXAMPLES

The following examples are presented for exemplary purposes only. Assuch, they are to be construed as representing embodiments or aspects ofthe present invention, and not to be construed as being limitingthereof.

Example 1

This example shows inhibition of PGHS-1 by N-dimethyl pyridinol andN-dimethyl pyrimidinol analogues of acetaminophen (ApAP). See FIG. 1.Hematin-reconstituted PGHS-1 5.4 nM in Tris HCl 100 mM, pH 8.0, 500 μMphenol, was pre-incubated with acetaminophen (ApAP),2-(dimethylamino)pyrimidin-5-ol (DM-Pym) or6-(dimethylamino)pyridin-3-ol (DM-Pyr) at varying concentrations at 37°C. for 20 minutes. At this time the reaction was initiated by additionof 0.5 μM of [14C] arachidonic acid. After 8 seconds, the reaction wasstopped by adding an ice cold mixture of diethyl ether/methanol/4.0 Mcitric acid. PGHS-1 activity was expressed as percent oxidation ofarachidonic acid compared with control where no inhibitor was added.Each data point represents the mean±S.E.M of six values.

Example 2

This example shows the effect of ApAP and DM-Pym analog on the initialrate of reduction of ferryl-myoglobin. See FIG. 2. Reduction from ferrylto ferric myoglobin by acetaminophen (ApAP, dashed line) or by2-(dimethylamino)pyrimidin-5-ol (DM-Pym, plain line) was monitored byrecording visible spectra between 350 and 650 nm. Ferryl myoglobin wasgenerated by incubating ferric myoglobin 10 μM in PBS with 10 μMhydrogen peroxide until there was no more change in the peak at 421 nm.At this time, the analogs were added to the cuvette at the finalconcentrations indicated and spectra were recorded every 15 sec for 2min. The initial rate of reduction was calculated as the variation ofabsorbance at 409 nm in the first two minutes after adding the analogs.In this period of time the rates of reduction were linear for eachconcentration of analog.

Example 3

This example shows the selective inhibition of COX isoforms byacetaminophen analogs. Hematin-reconstituted COX-1 (5.4 nM) or COX-2(10.8 nM) in Tris HCl 100 mM, pH 8.0, 500 μM phenol, was pre-incubatedwith N-dimethyl pyrimidinol (DM, plain line) or N-dimethyl pyridinol(DR, dashed line) at varying concentrations at 37° C. for 20 minutes.See FIG. 3. At this time the reaction was initiated by addition of 0.5μM of [14C] arachidonic acid. After 8 seconds, the reaction was stoppedby adding an ice cold mixture of diethyl ether/methanol/4.0 M citricacid. PGHS-1 activity was expressed as percent oxidation of arachidonicacid compared with control where no inhibitor was added. Each data pointrepresents the mean±S.E.M of six values.

Example 4

This example shows cytotoxicity of acetaminophen analogs in HepG2 cells.See FIG. 4. HepG2 were plated in multiple 96-well plates at 2×104 cellsper well and pretreated with vehicle only (0 μM inhibitors), ordifferent concentrations of acetaminophen (ApAP),2-(dimethylamino)pyrimidin-5-ol (DM-Pym) or6-(dimethylamino)pyridin-3-ol (DM-Pyr),2-(dimethylamino)-4,6-dimethylpyrimidin-5-ol (TM-Pym) or6-(dimethylamino)-2,4-dimethylpyridin-3-ol (TM-Pyr). Cell viabilityafter 24 h was determined by measuring ATP levels by luminescence assay.

Example 5

This example shows the effect of ethanol on cytotoxicity ofacetaminophen analogs in HepG2 cells. See FIG. 5. HepG2 were plated inmultiple 96-well plates at 2×104 cells per well and pretreated withethanol 100 μM for 2 hours. At this time, vehicle (0 μM inhibitors), oracetaminophen (ApAP), 2-(dimethylamino)pyrimidin-5-ol (DM-Pym) or6-(dimethylamino)yridine-3-ol (DM-Pyr),2-(dimethylamino)-4,6-dimethylpyrimidin-5-ol (TM-Pym) or6-(dimethylamino)-2,4-dimethylpyridin-3-ol (TM-Pyr) were added at 2 mMfinal concentration. Cell viability after 24 h was determined bymeasuring ATP levels by luminescence assay.

Example 6

This example features three examples of the present invention withstructures designed to modulate the donor characteristics of the 6-aminogroup, similar to acetaminophen, and fix the geometry of ringsubstituents by the constraints of a fused ring.

A similar structural motif can be found in uric acid, which isresponsible for more than half of human blood plasma antioxidantcapacity 25. Thus, a “urea bridge” has been chosen to protect the freeamine. This structural feature was intended to modulate the IP value ofthese novel analogs based on metabolic instability and cyto-toxicityshown by other embodiments of the present invention

Since the presence or the absence of C(2) and C(4) substituents in theheterocyclic phenol ring did not drastically alter pyridines andpyrimidines toxicity profile both analogs 12 and 13 have been prepared.The urethane analog 14 of the urea cycle 13 was also prepared.

The previously known 2-amino-5-bromo-nicotinonitrile (15) 26 has beenprepared from commercially available 2-amino-nicotinonitrile via anefficient nuclear monobromination, by treatment with N-bromosuccinimide(NBS) in the presence of a catalytic amount of NH4OAc 27. Reduction bythe borane-tetrahydrofuran complex (BH3-THF) followed by acidicdeprotection of the resulting borane furnished the diamino compound (16)which was converted to the cyclic urea (17) by action of1,1′-Carbonyldiimidazole (CDI). Tert-Butyloxycarbonyl (Boc) protectionof exchangeable amide protons is important for success of the Pdcatalyzed borylation reaction-oxidation two-step sequence 28. Therefore,the Boc protected cyclic urea (18) was reacted bis-(pinacolato)diboronand the crude product was oxidized giving rise to a separable mixture ofmono and di protected phenols (19 and 20). Either mono or di protectedphenols (or their mixture) can be deprotected by the reaction withmethanolic solution of hydrochloric acid followed by recrystallization.

The synthesis of analogs 13 and 14 originated from commerciallyavailable vitamin B6, which was converted to compound 21 by a highlyefficient two-step sequence 18. Bromination with concentratedhydrobromic acid 29 afforded crude 22, which was converted to compound23 by reaction with sodium azide. Diazo-substitution furnished compound24. Both diazo and azido groups were simultaneously reduced bypalladium-on-carbon catalyzed hydrogenation. Diamine 25 was converted topyridinol-fused ring analog 13 via CDI induced cyclic urea formation.Analog 14 was produced from compound 21, which was converted to6-amino-5-(hydroxymethyl)-2,4-dimethylpyridin-3-ol by a known procedure30 followed by protection with benzyl chloroformate (CbzCl) andsubsequent cyclization under the basic conditions.

The analogs if this example have been evaluated for their microsomalstability and cellular toxicity as described in the experimentalsection. Analogs 12, 13 and 14 have displayed a toxicity and stabilityprofile, which is consistent with our structure-to-biological propertyrelationship hypothesis. These compounds have no direct cytotoxicity atconcentrations below the millimmolar range. Their EC50 values forcytotoxicity are 5 to more than 10 times higher than that ofacetaminophen (Table 1). Three compounds were metabolized by human livermicrosomes to a lesser extent than acetaminophen, suggesting that invivo metabolism by microsomes would be less.

TABLE 1 Stability and direct cytotoxicity of novel generation ofpyridinols Microsomal stability Cellular Toxicity % left (EC₅₀, μM ± SD)ApAP  65 ± 12 594 ± 147 12 158 ± 33 4760 ± 36   13 87 ± 7 2965 ± 284  1497 ± 3 >5000

Microsomal stability is expressed as % of unmodified compound remainingafter reaction. Cellular toxicity is expressed as the con-centrationcausing a 50% decrease in total cellular ATP levels. Values representmeans±S.E.M.

Next, the efficiency of ApAP analogs of the present invention to inhibithemeprotein-catalyzed lipid oxidation were tested using COX-1 andmyoglobin. As shown in Table 2, examples of the present invention areable to inhibit myoglobin-induced arachidonic oxidation within the sameorder of magnitude, with analog 14 being the least potent. In contrary,analog 14 is most potent in inhibiting COX-1 activity followed by ApAPand analog 12. Interestingly, analog 13 was not able to significantlyinhibit COX-1 at concentrations up to 1 mM.

TABLE 2 Inhibition by ApAP of hemeprotein-catalyzed oxidation ofarachidonic acid. Analog ApAP #12 #13 #14 Mb 2.3 ± 0.2 ^(a)  1.2 ± 0.12.3 ± 0.4  5.3 ± 0.1 (μM ± SEM) COX-1 372^(b)  451 ± 35 >1,000  198 ± 25(μM ± SEM)

The inhibition of the lipid peroxidation produced by myoglobin (Mb) andCOX-1 by the different analogs was tested. Values represent the IC50 foreach hemeprotein and are expressed as means±S.E.M. a: value reportedfrom (1.). b: value reported from (3).

Oxidation of Oxidation of AA Structure AA by Mb by COX-1 (bicycliccompounds) IC₅₀ IC₅₀

1.15 ± 0.3 451 μM

2.29 ± 0.6 >1 mM

5.34 ± 0.1 μM 198.1 ± 50.1 μM

In conclusion, we investigated a number of potent phenolic heterocycleswith respect to their efficiency in inhibiting hemeprotein-catalyzedlipid oxidation and also their metabolic stability and toxicity. Severalimportant structure—stability and structure—toxicity relationshipstudies were used for the design of novel heterocyclic acetaminophenanalog series. Our results indicate that two analogs (12 and 14) mayrepresent a good alternative to acetaminophen with a similar efficiencyand better cytotoxicity profile. These analogs are promising candidatesfor studies in animal models of hepatotoxicity to determine whether theyrepresent lead compounds for development of drugs that could replaceacetaminophen.

The invention thus being described, it will be apparent to those skilledin the art that various modifications and variations can be made in thepresent invention without departing from the scope or spirit of theinvention. Other embodiments of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. Particularly, it should be obviousthat the embodiments described can be modified without departing fromthe spirit of the present invention.

Throughout this disclosure, various publications are referenced. Allreferences cited herein are expressly incorporated herein by referencein their entirety and are considered to be part of this disclosure. Ofcourse, all attachments submitted with the Specification areincorporated herein by reference in their entirety and are intended tobe considered part of the present patent application.

1. A method for treating, preventing, or reducing oxidative damage in amammalian subject comprising administering a therapeutically effectiveamount of a compound of the following formula:

wherein X is independently N or C, with the C being unsubstituted orsubstituted with H or alkyl; R₁ is independently H or alkyl; R₂ is H oralkyl; R₃ is H or alkyl R₄ is independently H or alkyl, or forms a6-member ring with R₂ or R₃ that contains C or O as ring members, the Cring members being unsubstituted or substituted with H or alkyl; or apharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the therapeutically effective amount is a heme-induced lipidperoxidation inhibiting amount.
 3. The method of claim 1, wherein thecompound is selected from the group consisting of:

wherein R₁ is H or alkyl; R₂ is H or alkyl; R₃ is H or alkyl; or apharmaceutically acceptable salts thereof.
 4. The method of claim 1,wherein the compound is:

or a pharmaceutically acceptable salts thereof.
 5. (canceled)
 6. Amethod of preventing or treating isoprostane-mediated tissue damage in amammalian subject comprising administering a therapeutically effectiveamount of a compound of the following formula:

wherein X is independently N or C, with the C being unsubstituted orsubstituted with H or alkyl; X₂ is C, O, or NR₅; R₁ is independently Hor alkyl; R₂ is H or alkyl; R₃ is H or alkyl R₄ is independently H oralkyl, or forms a 6-member ring with R₂ or R₃ that contains C or O asring members, the C ring members being unsubstituted or substituted withH or alkyl; R₅ is selected from hydrogen, alkyl, C₃₋₁₀ cycloalkyl, aryl,benzyl, heteroaryl, halogen, CN CF₃, CO, CO-alkyl; R₆ is selected fromhydrogen, alkyl, C₃₋₁₀ cycloalkyl, aryl, benzyl, heteroaryl, halogen, CNCF₃, CO, CO-alkyl; or a pharmaceutically acceptable salt thereof.
 7. Themethod of claim 6, wherein the therapeutically effective amount is anisoprostane synthesis inhibiting amount.
 8. The method of claim 6,wherein the compound is selected from the group consisting of

wherein R₁ is independently H or alkyl; R₂ is independently H or alkyl;R₃ is H or alkyl; or a pharmaceutically acceptable salts thereof.
 9. Themethod of claim 6, wherein the compound is:

or a pharmaceutically acceptable salts thereof.
 10. (canceled)
 11. Amethod for inhibiting cyclooxygenase or prostaglandin H synthaseenzymes, comprising administering a therapeutically effective amount ofa compound of the following formula:

wherein X is independently N or C, with the C being unsubstituted orsubstituted with H or alkyl; X₂ is C, O, or NR₅; R₁ is independently Hor alkyl; R₂ is H or alkyl; R₃ is H or alkyl R₄ is independently H oralkyl, or forms a 6-member ring with R₂ or R₃ that contains C or O asring members, the C ring members being unsubstituted or substituted withH or alkyl; R₅ is selected from hydrogen, alkyl, C₃₋₁₀ cycloalkyl, aryl,benzyl, heteroaryl, halogen, CN CF₃, CO, CO-alkyl; R₆ is selected fromhydrogen, alkyl, C₃₋₁₀ cycloalkyl, aryl, benzyl, heteroaryl, halogen, CNCF₃, CO, CO-alkyl; or a pharmaceutically acceptable salt thereof. 12.The method of claim 11, wherein the compound is selected from the groupconsisting of

wherein R₁ is independently H or alkyl; R₂ is independently H or alkyl;R₃ is H or alkyl; or a pharmaceutically acceptable salts thereof. 13.The method of claim 11, wherein the compound is:

or a pharmaceutically acceptable salts thereof. 14.-20. (canceled) 21.The method of claim 1, wherein the compound is selected from the groupconsisting of:


22. The method of claim 6, wherein the compound


23. The method of claim 11, wherein the compound


24. A method for treating, preventing, or reducing oxidative damage in amammalian subject comprising administering a therapeutically effectiveamount of a compound of the following formula:

wherein: X is N or C, with the C being unsubstituted or substituted withH or alkyl; R₁ is H or alkyl; R₂ is H or alkyl; R₃ is H or alkyl; R₄ isindependently H or alkyl; X₁ is

wherein R is H, alkyl, cycloalkyl, aryl, or —C(O)R; and Y is CH₂, NR, orO; or a pharmaceutically acceptable salt thereof.
 25. A method ofpreventing or treating isoprostane-mediated tissue damage in a mammaliansubject comprising administering a therapeutically effective amount of acompound of the following formula:

wherein X is N or C, with the C being unsubstituted or substituted withH or alkyl; R₁ is independently H or alkyl; R₂ is H or alkyl; R₃ is H oralkyl; R₄ is independently H or alkyl; and X₁ is

wherein R is H, alkyl, cycloalkyl, aryl, or —C(O)R; and Y is CH₂, NR, orO; or a pharmaceutically acceptable salt thereof.